Project Summary: Acylfulvenes, derivatives of illudin natural products, are a promising class of drugs for cancer treatment. They are notable for high therapeutic indices and selectivitiesfor tumor cells, and clinical trials are underway. Acylfulvenes are alkylating agents, but the detailed chemical mechanisms of DNA and protein-adduct formation, and how this relates to tumor-cell selectivity is not known. The long-term goals of this project are to define a mechanistic paradigm accounting for the selectivetoxicities of acylfulvenes that will lead to the development of combined-agent and tailored therapies designed to site-specifically tune acylfulvene toxicity. The objectivesof this application are to establish a link from acylfulvene bioactivation to biomolecular target modification and cytotoxicity. The specific aims are to (1) Characterize in vitro patterns of DNA alkylation by acylfulvenes and distinguish adducts that result from direct modification of DNA from those of bioactivation- coupled reactions; (2) Characterize the mechanism of inhibition of cellular redox proteins by acylfulvenes; (3) Determine the Influence of a bioactivating reductase on acylfulvene alkylation reactions and toxicity in cells. The research approach for aim 1 involves the synthesis of chemically activated molecular probes that will be used to identify products of DNA alkylation by bioactivatedacylfulvenes. We will use chemical characterization methods to identify the structures of acylfulvene DNA adducts. In aim 2, we will use enzyme-inhibition studies combined with a mass-spectrometry-based approach to structurally map protein covalent modification. In aim 3, we will use reductase-overexpressingcells and stable tumor cell lines to test the impact of adduct formation in the complex environment of the cell. Relevance: Acylfulvenes are a new class of drugs that kill cancer cells by becoming covalently linked to biomolecules in the cell and initiating a sequence of biological events that result in cell death. The initial chemical transformations that govern this process and how they differ in cells that are sensitive to acylfulvenes versus those that are not are unknown. The ability to selectivelytarget tumor cells is a central problem in cancer therapy and consequently understanding this mechanism is important for designing safer cancer therapy strategies.